THOMASON, KL; TOWLE, DW; HENRY, RP; Auburn Univ.; Mt. Desert Island Biol. Lab.; Aburn Univ.: Quantitative expression of carbonic anhydrase mRNA in the gills of Carcinus maenas during acclimation from high to low salinity
Carbonic anhydrase (CA) activity is known to be induced up to 12 fold in the posterior, ion transporting gills of the euryhaline green crab during acclimation from high (32 ppt) to low (10 ppt) salinity. CA induction takes place over the course of 4-7 days, suggesting that the process involves the synthesis of new enzyme. This process, however, has never been quantitatively examined. Using quantitative PCR, the levels of CA mRNA expression in the gills of the green crab were measured during the time course of acclimation from 32 to 10 ppt salinity. Total RNA was extracted from anterior and posterior gills and was reverse transcribed into cDNA. Gene specific primers for the nucleotide sequence of CA from the green crab were used with the cDNA in quantitative PCR (Stratagene MX 4000) to measure changes in the expression of CA mRNA. For crabs acclimated to 32 ppt, CA mRNA expression was relatively low and uniform in anterior and posterior gills (Ct values of 25.4 + 2.0 and 24.2 + 0.5 (N=5), respectively). At 24 hr after transfer to 10 ppt, the Ct value for posterior gills dropped to 22.6 + 0.7 and remained between 20 and 22 for the remainder of the 7 day time course. This represents an approximate 6 fold increase in CA mRNA expression. Ct values for anterior gills remained at approximately 25 over the initial 48 hr post-transfer and then varied between 23 and 25 between 72 hr and 7days post-transfer. Protein-specific CA activity in posterior gills doubled at 48 hr post-transfer and continued to increase through 96 hr after low salinity transfer. CA activity in anterior gills did not change. These results show that induction of CA activity is a result of the synthesis of new protein, which is due to gene activation and increased expression of CA mRNA. Supported by NSF IBN 02-3005 and an Auburn Univ. undergraduate research fellowship.
